Determination of β-Amyloid Peptide Signatures in Cerebrospinal Fluid

Mar 10, 2006 - Synopsis. We here report the use of immunoprecipitation in combination with MALDI-TOF MS to perform a detailed characterization of N- a...
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Determination of β-Amyloid Peptide Signatures in Cerebrospinal Fluid Using Immunoprecipitation-Mass Spectrometry Erik Portelius,* Ann Westman-Brinkmalm, Henrik Zetterberg, and Kaj Blennow Institute of Clinical Neuroscience, Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, 43180 Mo¨lndal, Sweden Received December 22, 2005

Abstract: Early pathogenic events in Alzheimer’s disease (AD) involve increased production and/or reduced clearance of β-amyloid (Aβ), especially the 42 amino acid fragment Aβ1-42. The Aβ1-42 peptide is generated through cleavage of the amyloid precursor protein by βand γ-secretase and is catabolised by a variety of proteolytic enzymes such as insulin-degrading enzyme and neprilysin. Here, we describe a method that employs immunoprecipitation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to determine the pattern of C-terminally truncated Aβ peptides in cerebrospinal fluid (CSF). Using antibodies coupled to magnetic beads, we have detected 18 C-terminally and 2 N-terminally truncated Aβ peptides in CSF. By determining the identity and profile of the truncated Aβ peptides, more insight may be gained about differences in the metabolism and structural properties of Aβ in AD. Finally, the Aβ fragment signatures may prove useful as a diagnostic test for AD. Keywords: Alzheimer’s disease • immunoprecipitation • mass spectrometry • MALDI-TOF MS • cerebrospinal fluid • β-amyloid

Introduction Alzheimer’s disease (AD) is one of the major neuropsychiatric disorders, affecting more than 20 million individuals worldwide. Senile plaques and neurofibrillary tangles throughout cortical and limbic regions of the brain are the two neuropathological hallmarks of the disease.1 The major component of senile plaques is β-amyloid (Aβ) that is generated from amyloid precursor protein (APP) by enzymatic cleavage involving β- and γ-secretase activities (Figure 1).2 Dysregulation of this process is believed to be an early or even pathogenic event in the course of the disease.2 Cleavage of APP by β-secretase generates an approximately 100 kDa soluble N-terminal fragment and a 12 kDa C-terminal fragment (C99). C99 can be further cleaved by γ-secretase to yield the APP intracellular domain (AICD) and different Aβ peptides (Figure 2A) of which the 42 amino acid fragment is the most hydrophobic and fibrillogenic (the amyloidogenic pathway).3 Another enzymatic activity, R-secretase, cuts APP in the middle of the Aβ region between amino acids * To whom correspondence should be addressed. E-mail: erik.portelius@ neuro.gu.se.

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16 and 17, thereby precluding production of Aβ peptides (the nonamyloidogenic pathway). The R-secretases identified so far all belong to the ADAM (a disintegrin and metalloprotease) family of proteins.4 Aβ1-40 and Aβ1-42 can be reproducibly measured in various biological fluids using different antibodies directed against their respective C-terminus.5 Other proteolytically processed Aβ peptides, however, are difficult to detect using standard methods, possibly because they comprise a heterogeneous set of both N- and C-terminally truncated peptides, some of which are present only at low levels. Several studies have shown that different Aβ-degrading proteases generate truncated Aβ fragments.6,7 One of these proteases, insulindegrading enzyme (IDE), has at least 6 cleavage sites within the Aβ-sequence (see Figure 2B).8-10 Other peptidases implicated in the Aβ catabolism include neprilysin (NEP), endothelin-converting enzymes (ECE-1 and ECE-2) and plasmin.11-16 We have earlier used various immunoassays for measurement of Aβ peptides in both plasma and cerebrospinal fluid (CSF).17-19 However, these techniques are limited in that they cannot detect and identify modifications of peptides (such as point mutations, adduction, phosphorylation, or oxidation). Moreover, they only detect Aβ fragments against which the antibodies were directed. Mass spectrometry, on the other hand, allows for detection of a variety of modified and truncated Aβ peptides, thus enabling a more detailed and unbiased analysis of fragments that may play a role in AD. Many studies have used mass spectrometry for studying Aβ peptides. Using antibodies covalently bound as an affinity matrix and matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS), it was found that there is a heterogeneous set of both C- and N-terminally truncated Aβ peptides in CSF with Aβ1-40 being the most abundant.20 Immunoprecipitation in combination with mass spectrometry (IP-MS) has been used for determination of the Aβ peptide profile in cultured cell media. By using 2 different antibodies, both N- and C-terminally truncated Aβ peptides were detected with MALDI-TOF MS.21 Others have used surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for analyzing CSF and found 11 C-terminally truncated Aβ peptides in total.22,23 These data indicate that truncated Aβ peptides might play an important role in the diagnostics for AD. However, the relatively low mass accuracy of SELDI-TOF MS (mass deviation between 300 and 600 ppm was reported for Aβ1-42) makes unambiguous identification of the peptides difficult. Furthermore, the Aβ 10.1021/pr050475v CCC: $33.50

 2006 American Chemical Society

technical notes

Portelius et al.

Figure 1. Metabolism of APP with Aβ generation. APP is a transmembrane protein with a large N-terminal extracellular tail. The largest isoform of APP (APP770) is shown. The β-amyloid (Aβ) domain, is partly embedded in the plasma membrane, and includes the first 28 residues just outside the membrane, and the first 12-14 residues in the transmembrane domain. APP can be processed along two main pathways: (A) In the R-secretase pathway, R-secretase cleaves APP within the Aβ domain, releasing the large soluble APP fragment (R-sAPP). The remaining C-terminal fragment (CTF), R-CTF or C83, is cleaved by the γ-secretase complex releasing the short p3 peptide. The remaining APP intracellular domain (AICD) is released into the cytoplasm. Since APP cleavage by R-secretase is within the Aβ domain this precludes Aβ generation. (B) In the β-secretase pathway, β-secretase cleaves APP just before the Aβ domain, releasing the soluble βsAPP. The remaining CTF, β-CTF or C99, is cleaved by the γ-secretase complex releasing the free 40 to 42 amino acid Aβ peptide. The remaining AICD is released into the cytoplasm.

fragments reported in the two different studies were not identical, even though they used the same antibody (6E10), indicating that there could be more C-terminally truncated Aβ peptides in CSF which were not detected. MALDI-TOF MS is a robust and sensitive method with much better mass accuracy in the lower peptide mass range than SELDI-TOF MS. We here report the use of immunoprecipitation in combination with MALDI-TOF MS to extract and perform a detailed characterization of truncated Aβ peptides in CSF. By determining the identity and profile of the truncated Aβ peptides in CSF more insight may be gained about differences in the metabolism and structural properties of Aβ in AD and normal aging.

Experimental Section Cerebrospinal Fluid. CSF samples were obtained from the Clinical Neurochemical Laboratory, Sahlgrenska University Hospital. The investigation was performed on CSF samples from patients who underwent lumbar puncture to exclude infectious disorders of the CNS. The inclusion criteria were normal white cell count and blood-brain barrier function, and absence of intrathecal IgG and IgM production. Patient identifications were removed and all samples were coded prior to analysis. The first 10-12 mL of CSF was collected and gently mixed to avoid possible gradient effects. The CSF samples were

centrifuged at 1800 × g for 10 min to eliminate cells and other insoluble material and kept at -20 °C until analysis (within 3-4 days). Immunoprecipitation. An aliquot (4 µg) of the monoclonal antibodies 6E10 (1 mg/mL, epitope 4-9), 4G8 (1 mg/mL, epitope 18-22), or 11A50-B10 (0.5 mg/mL, reactive to the C-terminus) (Signet Laboratories, Inc.), was separately added to 50 µL magnetic Dynabeads (Sheep anti mouse, IgG) and incubated overnight on a rocking platform at +4 °C. The remaining unbound antibody was removed by washing twice with phosphate-buffered saline (PBS, pH 7.4). After adding 1 mL CSF to the antibody-coated beads, the incubation was continued for an additional 1 h at +4 °C. The beads were pelleted for 5 min by using a magnetic particle concentrator (Dynal MPC) and washed twice with PBS (pH 7.4) and twice with 50 mM ammonium bicarbonate (pH 7.3). After the final wash, the extracted Aβ peptides were eluted by adding 20 µL 0.5% formic acid (FA) in water. After vortexing for 2 min in room temperature, the beads were pelleted using the magnetic particle concentrator and the supernatant was collected. The collected supernatant was dried down in a vacuum centrifuge and redissolved in 5 µL 0.1% FA in 20% acetonitrile (ACN). All solvents used were of HPLC quality and all aqueous solutions were made using 18.2 MΩ deionized water obtained from a Millipore purification system. Journal of Proteome Research • Vol. 5, No. 4, 2006 1011

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β-Amyloid Peptide Signatures in CSF

Figure 2. Aβ1-42 amino acid sequence with potential cleavage sites for (A) R-, β-, and γ-secretase and (B) 4 proteases marked (arrows above the sequence). Arrows bellow the sequence indicate the C-terminally truncated Aβ peptides immunoprecipitated from CSF in this study. I ) insulin-degrading enzyme, N ) neprilysin, E ) endothelin-converting enzyme (ECE-1, ECE-2), and P ) plasmin.

Mass Spectrometric Analysis of Immunoprecipitated Aβ. MALDI samples were prepared with the seed layer method.24 Briefly, a seed layer was created on a MALDI-TOF MS stainless steel sample probe (Bruker Daltonics Inc.) by depositing 0.5 µL (1 g/L) of alfa-cyano-4-cinnamic acid (CHCA, Fluka) dissolved in ACN. One microliter of saturated (15 g/L) CHCA in 0.1% trifluoroacetic acid in ACN/water (1:1 v/v) was added to an equal volume of the dissolved peptides and mixed. One microliter matrix/peptide solution was added to the probe and the sample was left to dry completely in air. MALDI-TOF MS measurements were performed using an AUTOFLEX instrument (Bruker Daltonics Inc.) operating in reflecting mode at 19 kV acceleration voltage. The spectra represent an average of 900 shots and were recorded up to 4600 Da. The spectra were calibrated using internal calibration (m/z 1826.8, 2068.0, 4130.0, and 4328.2) and each sample was analyzed in duplicate (except where noted). All mass spectra were analyzed using Bruker Daltonics flexanalysis 2.4, baseline subtracted and then smoothed with a 5-point Savitsky-Golay smooth. All reported m/z are the monoisotopic peak of the protonated molecule [M+ H]+ (except were noted). The sequence of Aβ peptides best matching the molecular mass obtained with MALDI-TOF MS was evaluated with an in-house developed software (PeptideMassCalculator).

Results and Discussion At least 18 different C-terminally truncated Aβ peptides were identified using IP with magnetic beads and MALDI-TOF MS analysis. Mass detection showed high accuracy and corresponded well to calculated peptide masses (better than 15 ppm deviation except Aβ1-18, Aβ1-20, and Aβ1-42, see Table 1). The 18 different C-terminally truncated Aβ peptides were detected in all of the CSF samples analyzed (4 different patients, see Figures 3 and 6A-C for mass spectra from 2 different patients). To determine the reproducibility of the method CSF from one of the patients was divided into 8 tubes and immunoprecipitated using 6E10 as described earlier. Each tube 1012

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Table 1. C-Terminally Truncated Aβ Peptides in CSFa C-terminally truncated peptide

measured massa (Da)

theoretical massa (Da)

deviationb (ppm)

1-12 1-13 1-14 1-15 1-16 1-17 1-18 1-19 1-20 1-28 1-30 1-33 1-34 1-37 1-38 1-39 1-40 1-42

1424.60 1561.68 1698.73 1826.78 1954.89 2067.97 2166.99 2314.11 2461.04 3261.50 3389.61 3672.77 3785.86 4072.98 4130.01 4229.07 4328.17 4512.21

1424.61 1561.67 1698.73 1826.78 1954.88 2067.96 2167.03 2314.10 2461.17 3261.54 3389.59 3672.78 3785.87 4073.00 4130.02 4229.09 4328.16 4512.28

7.02 6.40 0.00 0.00 5.12 4.84 18.5 4.32 52.8 12.3 5.90 2.72 2.64 4.91 2.42 4.73 2.31 15.5

a The masses presented are the monoisotopic protonated molecules [M+ H]+. b Mass deviation ) (mass measured - theoretical mass)/theoretical mass.

was analyzed in triplicate. Intensity ratios between Aβ1-40 and Aβ1-38 and Aβ1-17 and Aβ1-15, respectively, were used to calculate coefficients of variation (CV). The CV values were 10.7% for the Aβ1-40/Aβ1-38 ratio and 14.3% for the Aβ117/Aβ1-15 ratio. As seen in Figure 3, the 3 most intense peaks had m/z 4328.2, 2068.0, and 4130.0, corresponding to the masses of Aβ1-40, Aβ1-17, and Aβ1-38, respectively (see Table 1 for all peptides found). This is consistent with Aβ1-40 being the most abundant C-terminally truncated Aβ peptide in CSF.20 Aβ1-42 (m/z 4512.2) is known to have a much lower concentration and has also much lower signal-to-noise ratio (S/N) in the mass spectrum. However, the ratio between the Aβ1-42 and Aβ140 peaks in the mass spectrum cannot be interpreted as a direct reflection of their relative abundance in CSF since the ioniza-

technical notes

Figure 3. Mass spectrum showing C-terminally truncated Aβ peptides immunoprecipitated from CSF using the antibody 6E10 (Aβ40 in spectrum ) Aβ1-40). The peptides were eluted from the magnetic beads with 0.5% FA and the spectra represent an average of 900 shots.

Figure 4. MALDI-TOF MS mass spectrum showing peaks at m/z corresponding to the isotopic pattern of the Aβ1-42 peptide. The peak at m/z 4512.2 is the monoisotopic peak (peptides only containing 12C atoms) of the protonated molecule [M+ H]+.

tion efficiency might be different for the two peptides and since Aβ1-42 is more hydrophobic and less soluble than Aβ1-40. This argument is also true for other fragments found in this study. The peak corresponding to Aβ1-42 had a S/N ratio below 5 but the measured mass, which deviated only 15.5 ppm from the theoretical mass, verified the Aβ1-42 identity (Figure 4). It should also be noted that different Aβ peptides may differ in protein interaction capacity and active or passive transport

Portelius et al.

Figure 5. MALDI-TOF MS mass spectrum showing peaks corresponding to the protonated molecule Aβ1-18 [M + H]+ and the doubly protonated molecule of Aβ1-40 [M + 2H]2+. The isotopic pattern of both peptides is resolved.

Figure 6. Mass spectra showing C- and N-terminally truncated Aβ peptides immunoprecipitated from CSF using the antibodies A) 6E10, B) 4G8 and C) 11A50-B10. The m/z 2037.5, 2066.3, and 2165.7 corresponds to the second charge state of Aβ1-37 [Aβ137 + 2H]2+, Aβ1-38 [Aβ1-38 + 2H]2+, and Aβ1-40 [Aβ1-40 + 2H]2+, respectively.

from brain tissue into CSF. Thus, peak intensity might not directly reflect the activity of different Aβ degrading enzymes. The dominant low mass fragment was Aβ1-17, which is somewhat unexpected, since there is no known major cleavage site within the Aβ peptide at this position. The finding indicates that R-secretase may cleave at this position too, but it is also possible that another protease activity (e.g., ECE-1, see below) Journal of Proteome Research • Vol. 5, No. 4, 2006 1013

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is responsible for this particular fragment. According to the literature, R-secretase-mediated cleavage generates an Aβ1740/42 peptide. Thus, there should be a fragment corresponding to Aβ1-16. Surprisingly, the intensity of the 1-16 fragment was markedly lower than that of Aβ1-17. One possible explanation is that Aβ1-16 generated by R-secretase may be processed further to Aβ1-15, which has been previously described.25 Two protease activities that could give the same fragment as R-secretase are ECE-111 and plasmin14,15 (see Figure 2B). ECE-1 has also been shown to cleave between amino acid 17-18 and 19-20, which might explain the peptides Aβ1-17 and Aβ1-19 (m/z 2068.0 and 2314.1, respectively) found in this study. However, if ECE-1 were solely responsible for the abundant Aβ1-17 fragment, it would indicate that this protease is a major Aβ-degrading enzyme in humans which differs from data generated in transgenic mice suggesting this role is played by IDE.26 Aβ1-17, Aβ1-18, and Aβ1-19 may in part be generated by proteolysis of Aβ after γ-secretase-mediated Aβ release, but experiments have shown that some of these Aβ variants are also formed when γ-secretase is inhibited, indicating that they are likely to be products of alternative processing of the C99 fragment of APP by proteases such as R-secretases.27 Given the multitude of candidate R-secretases in the ADAM family, it seems likely that they may have somewhat differing specificities for cutting sites around amino acid 16 of Aβ although this remains to be proven. IDE has potential cleavage sites that could give C-terminally truncated peptides explaining most of the shorter peptides found in this study. For example, IDE cleaves between amino acid 12-13, 13-14, 14-15, 19-20, 20-21, and 28-29, which correlates well with the C-terminally truncated peptides found in this study (see Figure 2B). The only fragment that cannot be explained by known Aβdegrading protease activities at this stage is Aβ1-18 (m/z 2167.0). The m/z of Aβ1-18 [M + H]+ is almost identical to the doubled charged m/z of Aβ1-40 [M + 2H]2+ but two distinct isotopic patterns could be distinguished in the mass spectrum (see Figure 5). The Aβ1-18 fragment has earlier been described in CSF using SELDI-TOF MS, however, the mass deviation was 415 ppm.23 As seen in Figure 3, there is a small peak at m/z 2777.5 which could correspond to Aβ1-23. At this stage, however, its identity is uncertain due to high mass deviation (433.5 ppm) and low S/N (1.8). According to some reports, γ-secretase has as many as seven potential cleavage sites in the Aβ peptide generating the C-terminally truncated peptides Aβ1-33, Aβ1-34, Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42.28-30 All of these fragments were found in this study (see Figure 3). The activity of neprilysin (NEP) might explain the peptides Aβ1-28 and Aβ130 but they were only present at low concentrations (S/N was below 5 for the two peaks), although they both had a mass accuracy better then 13 ppm. Another fragment found, which could be explained by the activity of neprilysin, is Aβ1-19. An additional protease activity that could cleave between amino acid 28-29 besides neprilysin, generating Aβ1-28, is plasmin. To verify the C-terminally truncated Aβ peptides identified using the antibody 6E10, the antibodies 4G8 and 11A50-B10, epitope 18-22 and specific against the C-terminus of Aβ40, respectively, were employed in the immunoprecipitation experiments. Figure 6 shows the fragment pattern obtained from CSF from one patient using the three different antibodies. Notably, the fragment pattern obtained using 6E10 was highly 1014

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Figure 7. MALDI-TOF MS mass spectra showing peaks at m/z corresponding to the isotopic pattern of the N-terminally truncated peptide Aβ11-42 using the antibodies (A) 4G8 and (B) 11A50-B10.

similar to what was seen in a different patient (compare Figures 3 and 6A). The pattern was also similar using 4G8 with the exception that the shorter fragments were not detected. The latter result is expected since the 4G8 epitope includes amino acid 18-22. In total, 8 C-terminally truncated Aβ peptides were found (Aβ1-30, Aβ1-33, Aβ1-34, Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42) using antibody 4G8. Two different N-terminally truncated Aβ peptides were also detected, Aβ1130 (m/z 2212.1, mass deviation 2.2 ppm) and Aβ11-40 (m/z 3150.7, mass deviation 19.9 ppm, see Figures 6B and 7A). Experiments employing the antibody 11A50-B10 (reactive to the C-terminus of Aβ40) verified Aβ1-40 and Aβ11-40 (Figures 6C and 7B). Earlier studies have shown that BACE may cleave APP between Tyr and Glu at position 10-11 in the Aβ sequence.31 Furthermore, the Aβ11-30 fragment might indicate concerted cleavage by BACE and neprilysin, since the latter has a known cleavage site between amino acid 30-31 (Figure 2B). Altogether, the results obtained by the three different antibodies corroborate each other and also exclude the risk that the fragment patterns detected might be the result of unspecific degradation, e.g., due to formic acid-induced degradation in vitro. The method described here detected one Aβ peptide modification. Methionine, amino acid 35 in the Aβ sequence, is susceptible to oxidation, which would affect the C-terminally truncated Aβ peptides Aβ1-37, Aβ1-38, Aβ1-39, and Aβ140. An additional peak, which had the mass of the peptide plus 16 Da (see Figure 3), was found for all those fragments. As seen in Figures 2A-B, 3, and 6A, there is an 8-residue segment of the Aβ peptide (Ala21-Lys28) that seems resistant to proteolytic degradation. By using limited proteolysis, for identification of protease-resistant segments within the Aβ peptide, others have reported similar results.32,33 For determination of the Aβ1-40 peptide conformation when engaged in the amyloid fibril, one study used scanning proline mutagenesis and predicted a parallel β-helix folding motif in which each

technical notes Aβ(15-36) region is involved in a protected conformation. They also showed that residues 22, 23, 29, and 30 are involved in turns.34 They further investigated the structural properties of Aβ protofibrils and predict a relatively flexible loop, involving residues 22-29, spanning 2 β-structure segments.35 Another study indicate a loop in the region 24-28 forming a structure that is resistant to proteolysis.33 These data, in conjunction with the in vivo results presented here, suggest that the Ala21-Lys28 segment of Aβ may be protease-resistant either by its intrinsic structure or by homo- and/or heterotypic protein-protein interactions that may block recognition sites for proteolytic enzymes.

Conclusion Immunoprecipitation experiments employing three different anti-Aβ antibodies and mass spectrometric analysis of Aβ peptides from CSF revealed a distinct pattern of several N- and C-terminally truncated Aβ species. The information corroborates earlier in vitro data indicating that an 8-residue segment of the peptide, Ala21-Lys28, may form a protease-resistant structure. The pattern further deepens our understanding of how the Alzheimer-associated Aβ peptide is degraded in vivo in humans and points to the involvement of a multitude of protease activities that may protect humans from Aβ overload in the brain. Finally, Aβ fragment signatures could potentially be used as a diagnostic test to distinguish AD patients from nondemented controls and other dementia disorders, a hypothesis that will be tested in future investigations.

Acknowledgment. This work was supported by grants from the Swedish Medical Research Council (Project Nos. 14002 and 12575), the Swedish Council for Working Life and Social Research, the Sahlgrenska University Hospital, the Go¨teborg Medical Society, the Swedish Medical Society, the Swedish Society for Medical Research, Swedish Brain Power, Stiftelsen Gamla Tja¨narinnor, Magnus Bergvalls stiftelse, A° hlen stiftelsen, Fredrik & Ingrid Thurings stiftelse, Gun & Bertil Stohnes stiftelse and Alzheimer Foundation, Sweden. Gunnar Brinkmalm is warmly acknowledged for the development of the PeptideMassCalculator software. References (1) Tomlinson, B. E.; Blessed, G.; Roth, M. Observations on the brains of nondemented old people. J. Neurol. Sci. 1968, 7, 331-356. (2) Selkoe, D. J. Alzheimer’s disease: genes, proteins, and therapy. Physiol. Rev. 2001, 81 (2), 741-766. (3) Jarrett, J. T.; Berger, E. P.; Lansbury, P. T., Jr. The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer’s disease. Biochemistry 1993, 32 (18), 4693-4697. (4) Allinson, T. M.; Parkin, E. T.; Turner, A. J.; Hooper, N. M. ADAMs family members as amyloid precursor protein alpha-secretases. J. Neurosci. Res. 2003, 74 (3), 342-352. (5) Mehta, P. D.; Pirttila, T.; Mehta, S. P.; Sersen, E. A.; Aisen, P. S.; Wisniewski, H. M. Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease. Arch. Neurol. 2000, 57 (1), 100-105. (6) Iwata, N.; Higuchi, M.; Saido, T. C. Metabolism of amyloid-beta peptide and Alzheimer’s disease. Pharmacol. Ther. 2005, 108 (2), 129-148. (7) Eckman, E. A.; Eckman, C. B. Abeta-degrading enzymes: modulators of Alzheimer’s disease pathogenesis and targets for therapeutic intervention. Biochem. Soc. 2005, 33 (Pt 5), 1101-1105. (8) Mukherjee, A.; Song, E.; Kihiko-Ehmann, M.; Goodman, J. P., Jr.; Pyrek, J. S.; Estus, S.; Hersh, L. B. Insulysin hydrolyzes amyloid beta peptides to products that are neither neurotoxic nor deposit on amyloid plaques. J. Neurosci. 2000, 20 (23), 8745-8749. (9) Song, E. S.; Hersh, L. B. Insulysin: an allosteric enzyme as a target for Alzheimer’s disease. J. Mol. Neurosci. 2005, 25 (3), 201-206.

Portelius et al. (10) Chesneau, V.; Vekrellis, K.; Rosner, M. R.; Selkoe, D. J. Purified recombinant insulin-degrading enzyme degrades amyloid betaprotein but does not promote its oligomerization. Biochem. J. 2000, 351 Pt 2, 509-516. (11) Eckman, E. A.; Reed, D. K.; Eckman, C. B. Degradation of the Alzheimer’s amyloid beta peptide by endothelin-converting enzyme. J. Biol. Chem. 2001, 276 (27), 24540-24548. (12) Howell, S.; Nalbantoglu, J.; Crine, P. Neutral endopeptidase can hydrolyze beta-amyloid(1-40) but shows no effect on betaamyloid precursor protein metabolism. Peptides 1995, 16 (4), 647-652. (13) Leissring, M. A.; Lu, A.; Condron, M. M.; Teplow, D. B.; Stein, R. L.; Farris, W.; Selkoe, D. J. Kinetics of amyloid beta-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays. J. Biol. Chem. 2003, 278 (39), 3731437320. (14) Tucker, H. M.; Kihiko, M.; Caldwell, J. N.; Wright, S.; Kawarabayashi, T.; Price, D.; Walker, D.; Scheff, S.; McGillis, J. P.; Rydel, R. E.; Estus, S. The plasmin system is induced by and degrades amyloid-beta aggregates. J. Neurosci. 2000, 20 (11), 39373946. (15) Van Nostrand: W. E.; Porter, M. Plasmin cleavage of the amyloid beta-protein: alteration of secondary structure and stimulation of tissue plasminogen activator activity. Biochemistry 1999, 38 (35), 11570-11576. (16) Iwata, N.; Tsubuki, S.; Takaki, Y.; Shirotani, K.; Lu, B.; Gerard, N. P.; Gerard, C.; Hama, E.; Lee, H. J.; Saido, T. C. Metabolic regulation of brain Abeta by neprilysin. Science 2001, 292 (5521), 1550-1552. (17) Olsson, A.; Vanderstichele, H.; Andreasen, N.; De Meyer, G.; Wallin, A.; Holmberg, B.; Rosengren, L.; Vanmechelen, E.; Blennow, K. Simultaneous measurement of beta-amyloid(1-42), total tau, and phosphorylated tau (Thr181) in cerebrospinal fluid by the xMAP technology. Clin. Chem. 2005, 51 (2), 336345. (18) Hoglund, K.; Wiklund, O.; Vanderstichele, H.; Eikenberg, O.; Vanmechelen, E.; Blennow, K. Plasma levels of beta-amyloid(140), beta-amyloid(1-42), and total beta-amyloid remain unaffected in adult patients with hypercholesterolemia after treatment with statins. Arch. Neurol. 2004, 61 (3), 333-337. (19) Andreasen, N.; Hesse, C.; Davidsson, P.; Minthon, L.; Wallin, A.; Winblad, B.; Vanderstichele, H.; Vanmechelen, E.; Blennow, K. Cerebrospinal fluid beta-amyloid(1-42) in Alzheimer disease: differences between early- and late-onset Alzheimer disease and stability during the course of disease. Arch. Neurol. 1999, 56 (6), 673-680. (20) Vigo-Pelfrey, C.; Lee, D.; Keim, P.; Lieberburg, I.; Schenk, D. B. Characterization of beta-amyloid peptide from human cerebrospinal fluid. J. Neurochem. 1993, 61 (5), 1965-1968. (21) Wang, R.; Sweeney, D.; Gandy, S. E.; Sisodia, S. S. The profile of soluble amyloid beta protein in cultured cell media. Detection and quantification of amyloid beta protein and variants by immunoprecipitation-mass spectrometry. J. Biol. Chem. 1996, 271 (50), 31894-31902. (22) Lewczuk, P.; Esselmann, H.; Meyer, M.; Wollscheid, V.; Neumann, M.; Otto, M.; Maler, J. M.; Ruther, E.; Kornhuber, J.; Wiltfang, J. The amyloid-beta (Abeta) peptide pattern in cerebrospinal fluid in Alzheimer’s disease: evidence of a novel carboxyterminally elongated Abeta peptide. Rapid Commun. Mass Spectrom. 2003, 17 (12), 1291-1296. (23) Maddalena, A. S.; Papassotiropoulos, A.; Gonzalez-Agosti, C.; Signorell, A.; Hegi, T.; Pasch, T.; Nitsch, R. M.; Hock, C. Cerebrospinal Fluid Profile of Amyloid b Peptides in Patients with Alzheimer’s Disease Determined by Protein Biochip Technology. Neurodegen. Dis. 2004, 1 (4-5), 231-235. (24) Westman, A.; Karlsson, G.; Ekman, R. Comparison of MALDIMS sample preparation strategies for proteins in complex biological mixtures. Adv. Mass Spectrom. 1998, 14, 1-8. (25) Bradbury, L. E.; LeBlanc, J. F.; McCarthy, D. B. ProteinChip arraybased amyloid beta assays. Methods Mol. Biol. 2004, 264, 245257. (26) Leissring, M. A.; Farris, W.; Chang, A. Y.; Walsh, D. M.; Wu, X.; Sun, X.; Frosch, M. P.; Selkoe, D. J. Enhanced proteolysis of betaamyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron 2003, 40 (6), 1087-1093. (27) Vandermeeren, M.; Geraerts, M.; Pype, S.; Dillen, L.; Van Hove, C.; Mercken, M. The functional gamma-secretase inhibitor prevents production of amyloid beta 1-34 in human and murine cell lines. Neurosci. Lett. 2001, 315 (3), 145-148.

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β-Amyloid Peptide Signatures in CSF (28) Beher, D.; Wrigley, J. D.; Owens, A. P.; Shearman, M. S. Generation of C-terminally truncated amyloid-beta peptides is dependent on gamma-secretase activity. J. Neurochem. 2002, 82 (3), 563575. (29) Fluhrer, R.; Multhaup, G.; Schlicksupp, A.; Okochi, M.; Takeda, M.; Lammich, S.; Willem, M.; Westmeyer, G.; Bode, W.; Walter, J.; Haass, C. Identification of a beta-secretase activity, which truncates amyloid beta-peptide after its presenilin-dependent generation. J. Biol. Chem. 2003, 278 (8), 5531-5538. (30) Murphy, M. P.; Hickman, L. J.; Eckman, C. B.; Uljon, S. N.; Wang, R.; Golde, T. E. gamma-Secretase, evidence for multiple proteolytic activities and influence of membrane positioning of substrate on generation of amyloid beta peptides of varying length. J. Biol. Chem. 1999, 274 (17), 11914-11923. (31) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J. C.; Collins, F.; Treanor, J.; Rogers, G.; Citron, M. Beta-secretase cleavage

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of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 1999, 286 (5440), 735741. Kheterpal, I.; Williams, A.; Murphy, C.; Bledsoe, B.; Wetzel, R. Structural features of the Abeta amyloid fibril elucidated by limited proteolysis. Biochemistry 2001, 40 (39), 11757-11767. Lazo, N. D.; Grant, M. A.; Condron, M. C.; Rigby, A. C.; Teplow, D. B. On the nucleation of amyloid beta-protein monomer folding. Protein Sci. 2005, 14 (6), 1581-1596. Williams, A. D.; Portelius, E.; Kheterpal, I.; Guo, J. T.; Cook, K. D.; Xu, Y.; Wetzel, R. Mapping abeta amyloid fibril secondary structure using scanning proline mutagenesis. J. Mol. Biol. 2004, 335 (3), 833-842. Williams, A. D.; Sega, M.; Chen, M.; Kheterpal, I.; Geva, M.; Berthelier, V.; Kaleta, D. T.; Cook, K. D.; Wetzel, R. Structural properties of Abeta protofibrils stabilized by a small molecule. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (20), 7115-7120.

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